Constriction Region Research Articles

The contribution of porins to enterobacterial drug resistance.

In Enterobacteriaceae, susceptibility to cephalosporins and carbapenems is often associated with membrane and enzymatic barrier resistance. For about 20 years, a large number of Klebsiella pneumoniae, Escherichia coli and Enterobacter cloacae presenting ß-lactam resistance have been isolated from medical clinics. In addition, some of the resistant isolates exhibited alterations in the outer membrane porin OmpC-OmpF orthologues, resulting in the complete absence of gene expression, replacement by another porin or mutations affecting channel properties. Interestingly, for mutations reported in OmpC-OmpF orthologues, major changes in pore function were found to be present in the gene encoding for OmpC. The alterations were located in the constriction region of the porin and the resulting amino acid substitutions were found to induce severe restriction of the lumen diameter and/or alteration of the electrostatic field that governs the diffusion of charged molecules. This functional adaptation through porins maintains the entry of solutes necessary for bacterial growth but critically controls the influx of harmful molecules such as β-lactams at a reduced cost. The data recently published show the importance of understanding the underlying parameters affecting the uptake of antibiotics by infectious bacteria. Furthermore, the development of reliable methods to measure the concentration of antibiotics within bacterial cells is key to combat impermeability-resistance mechanisms.

Karyotypic description and comparison of Litoria (L.) paraewingi (Watson et al., 1971), L.ewingii (Duméril et Bibron, 1841) and L.jervisiensis (Duméril et Bibron, 1841) (Amphibia, Anura).

The karyotype of Litoria (L.) paraewingi (Watson et al., 1971) (Big River State Forest, Victoria) is described here for the first time. It is prepared following tissue culture of toe clipping macerates, cryopreservation, reculture and conventional 4',6-diamidino-2-phenylindole (DAPI) staining. The L.paraewingi karyotype is then compared to similarly processed IUCN (International Union for the Conservation of Nature) least concern members L.ewingii (Duméril et Bibron, 1841) (southern Victoria) and L.jervisiensis (Duméril et Bibron, 1841) (Myall Lakes National Park, New South Wales), all members of the same L.ewingii complex/group. The L.paraewingi diploid number is 2n = 26, the same as for the other two species. Litoriaparaewingi chromosomes 1, 2, 6 and 7 are submetacentric, chromosomes 3 and 5 are subtelocentric and the remainder are metacentric. No secondary constriction or putative nucleolus organiser region (NOR) was readily identifiable following conventional DAPI staining in any scored L.paraewingi metaphase spread. Conversely, a putative NOR was readily identifiable on the long arm of chromosome 1 in all examined metaphase spreads for the other two species. The karyotypes of L.ewingii and L.jervisiensis here further differ from L.paraewingi with chromosome 1 being metacentric and chromosomes 8 and 10 being submetacentric for both former species. The L.jervisiensis karyotype differs from those of L.ewingii and L.paraewingi by DAPI staining with: (i) apparent relative length inversion of subtelocentric chromosome 3 and metacentric chromosome 4 and (ii) chromosome 6 being metacentric rather than submetacentric. All three species have a highly conserved chromosome morphology with respect to chromosomes 2, 5, 7, 9, 11, 12 and 13. The greatest gross morphological difference karyotypically is observed between L.paraewingi and L.jervisiensis. These karyotype data support the previous phylogenetic separation of these three species based upon genetic compatibility and behavioural, biochemical and molecular genetic analyses.

Controlling DNA Fragments Translocation across Nanopores with the Synergic Use of Site-Directed Mutagenesis, pH-Dependent Charge Tuning, and Electroosmotic Flow.

Biological and solid-state nanopores are at the core of transformative techniques and nanodevices, democratizing the examination of matter and biochemical reactions at the single-molecule level, with low cost, portability, and simplicity in operation. One of the crucial hurdles in such endeavors is the fast analyte translocation, which limits characterization, and a rich number of strategies have been explored over the years to overcome this. Here, by site-directed mutagenesis on the α-hemolysin protein nanopore (α-HL), sought to replace selected amino acids with glycine, electrostatic binding sites were induced on the nanopore's vestibule and constriction region and achieved in the most favorable case a 20-fold increase in the translocation time of short single-stranded DNA (ssDNA) at neutral pH, with respect to the wild-type (WT) nanopore. We demonstrated an efficient tool of controlling the ssDNA translocation time, via the interplay between the nanopore-ssDNA surface electrostatic interactions and electroosmotic flow, all mediated by the pH-dependent ionization of amino acids lining the nanopore's translocation pathway. Our data also reveal the nonmonotonic, pH-induced alteration of ssDNA average translocation time. Unlike mildly acidic conditions (pH ∼ 4.7), at a pH ∼ 2.8 maintained symmetrically or asymmetrically across the WT α-HL, we evidenced the manifestation of a dominant electroosmotic flow, determining the speeding up of the ssDNA translocation across the nanopore by counteracting the ssDNA-nanopore attractive electrostatic interactions. We envision potential applications of the presented approach by enabling easy-to-use, real-time detection of short ssDNA sequences, without the need for complex biochemical modifications to the nanopore to mitigate the fast translocation of such sequences.

Structural and functional analysis of aquaporins in Bombus terrestris

Bombus terrestris are efficient pollinators in forestry and agriculture, with higher cold tolerance than other bees. Yet, their cold tolerance mechanism remains unclear. Aquaporins (AQPs) function as cell membrane proteins facilitating rapid water flow, aiding in osmoregulation. Recent studies highlight the importance of insect AQPs in dehydration and cold stress. Comparative transcriptome analysis of B. terrestris under cold stress revealed up-regulation of four AQPs, indicating their potential role in cold tolerance. Seven AQPs—Eglp1, Eglp2, Eglp3, DRIP, PRIP, Bib, and AQP12L—have been identified in B. terrestris. These are widely expressed in various tissues, particularly in the alimentary canal and Malpighian tubules. Functional analysis of BterAQPs in the Xenopus laevis oocytes expressing system showed distinct water and glycerol selectivity, with BterDrip exhibiting the highest water permeability. Molecular modeling of BterDrip revealed six transmembrane domains, two NPA motifs, and an ar/R constriction region (Phe131, His256, Ser265, and Arg271), likely contributing to its water selectivity. Silencing BterDRIP accelerated mortality in B. terrestris under cold stress, highlighting the crucial role of BterDRIP in their cold tolerance and providing a molecular mechanism for their cold adaptation.

Mechanosensitive channel MscL gating transitions coupling with constriction point shift.

The mechanosensitive channel of large conductance (MscL) acts as an "emergency release valve" that protects bacterial cells from acute hypoosmotic stress, and it serves as a paradigm for studying the mechanism underlying the transduction of mechanical forces. MscL gating is proposed to initiate with an expansion without opening, followed by subsequent pore opening via a number of intermediate substates, and ends in a full opening. However, the details of gating process are still largely unknown. Using in vivo viability assay, single channel patch clamp recording, cysteine cross-linking, and tryptophan fluorescence quenching approach, we identified and characterized MscL mutants with different occupancies of constriction region in the pore domain. The results demonstrated the shifts of constriction point along the gating pathway towards cytoplasic side from residue G26, though G22, to L19 upon gating, indicating the closed-expanded transitions coupling of the expansion of tightly packed hydrophobic constriction region to conduct the initial ion permeation in response to the membrane tension. Furthermore, these transitions were regulated by the hydrophobic and lipidic interaction with the constricting "hot spots". Our data reveal a new resolution of the transitions from the closed to the opening substate of MscL, providing insights into the gating mechanisms of MscL.

Regional centromere configuration in the fungal pathogens of the Pneumocystis genus.

Centromeres are constricted chromosomal regions that are essential for cell division. In eukaryotes, centromeres display a remarkable architectural and genetic diversity. The basis of centromere-accelerated evolution remains elusive. Here, we focused on Pneumocystis species, a group of mammalian-specific fungal pathogens that form a sister taxon with that of the Schizosaccharomyces pombe, an important genetic model for centromere biology research. Methods allowing reliable continuous culture of Pneumocystis species do not currently exist, precluding genetic manipulation. CENP-A, a variant of histone H3, is the epigenetic marker that defines centromeres in most eukaryotes. Using heterologous complementation, we show that the Pneumocystis CENP-A ortholog is functionally equivalent to CENP-ACnp1 of S. pombe. Using organisms from a short-term in vitro culture or infected animal models and chromatin immunoprecipitation (ChIP)-Seq, we identified CENP-A bound regions in two Pneumocystis species that diverged ~35 million years ago. Each species has a unique short regional centromere (<10 kb) flanked by heterochromatin in 16-17 monocentric chromosomes. They span active genes and lack conserved DNA sequence motifs and repeats. These features suggest an epigenetic specification of centromere function. Analysis of centromeric DNA across multiple Pneumocystis species suggests a vertical transmission at least 100 million years ago. The common ancestry of Pneumocystis and S. pombe centromeres is untraceable at the DNA level, but the overall architectural similarity could be the result of functional constraint for successful chromosomal segregation.IMPORTANCEPneumocystis species offer a suitable genetic system to study centromere evolution in pathogens because of their phylogenetic proximity with the non-pathogenic yeast S. pombe, a popular model for cell biology. We used this system to explore how centromeres have evolved after the divergence of the two clades ~ 460 million years ago. To address this question, we established a protocol combining short-term culture and ChIP-Seq to characterize centromeres in multiple Pneumocystis species. We show that Pneumocystis have short epigenetic centromeres that function differently from those in S. pombe.

Unleashing the Power of Plasma and Flow: A Novel Cold Plasma‐Oscillatory System for Enhanced Continuous Biodiesel Production from Sunflower Oil

While fossil fuel resources are limited, the increasing demand for energy in the industrial and transportation sectors has led to using renewable fuels. This study aims to improve biodiesel production from sunflower oil by combining cold plasma and oscillatory systems through a transesterification process. The four main operational variables are examined, such as molar ratio (MR) (1:6–1:12), catalyst concentration (CC) (0.5–1.25 wt%), plasma exposure time (PET) (25–75 s), and residence time (RT) (30–90 s). The smooth periodic constriction reactor's physical attributes, including the diameter of the tube (D), the diameter of the constricted region (d0), and the distance between baffles (l), as well as the properties of the cold plasma system, are determined. The data analysis presents that PET, RT, CC, and MR have the greatest effect on efficiency. This optimization indicates that by exposing the reaction mixture to plasma for 59.99 s, adding 1.09% by weight of the catalyst, and with a MR of 7.17 in 86.43 s, biodiesel with a conversion percentage of 94.75% is produced. The results show that the optimum point matches with EN 14214 standard.

Mechanism of hydrophobic gating in the acetylcholine receptor channel pore.

Neuromuscular acetylcholine receptors (AChRs) are hetero-pentameric, ligand-gated ion channels. The binding of the neurotransmitter acetylcholine (ACh) to two target sites promotes a global conformational change of the receptor that opens the channel and allows ion conduction through the channel pore. Here, by measuring free-energy changes from single-channel current recordings and using molecular dynamics simulations, we elucidate how a constricted hydrophobic region acts as a "gate" to regulate the channel opening in the pore of AChRs. Mutations of gate residues, including those implicated in congenital myasthenia syndrome, lower the permeation barrier of the channel substantially and increase the unliganded gating equilibrium constant (constitutive channel openings). Correlations between hydrophobicity and the observed free-energy changes, supported by calculations of water densities in the wild-type versus mutant channel pores, provide evidence for hydrophobic wetting-dewetting transition at the gate. The analysis of a coupled interaction network provides insight into the molecular mechanism of closed- versus open-state conformational changes at the gate. Studies of the transition state by "phi"(φ)-value analysis indicate that agonist binding serves to stabilize both the transition and the open state. Intersubunit interaction energy measurements and molecular dynamics simulations suggest that channel opening involves tilting of the pore-lining M2 helices, asymmetric outward rotation of amino acid side chains, and wetting transition of the gate region that lowers the barrier to ion permeation and stabilizes the channel open conformation. Our work provides new insight into the hydrophobic gate opening and shows why the gate mutations result in constitutive AChR channel activity.

Investigation of Erying–Powell fluid flow in the elliptical multi‐stenosed artery: Application of perturbation method via polynomial solutions

AbstractIn the current work, we analyzed the non‐Newtonian rheology of blood through a multi‐stenosed artery with a cross‐section of elliptical shape. The blood is regarded as Erying–Powell fluid, and flow is considered to have no slip at the stenotic wall. The mathematical model is processed to a non‐dimensional form, and conditions of mild stenosis are utilized to decrease its nonlinearity. The resulting equations are solved by applying the perturbation technique by considering the fluid characteristic parameter as the perturbation parameter. The solution is completed by using the polynomial of degree four. The solutions of mathematical equations are deeply examined by graphical analysis. The non‐Newtonian impacts are predominant in the surrounding of the stenosed wall along the minor axis of the elliptical artery. The height of stenosis affects the pressure rise and flow resistance. The shear stress at the arterial wall is very high in the stenotic region and has more potent effects in the neighboring boundary point on the minor axis. Progressive stenosis causes a reduction in the blood flow velocity surrounding the arterial wall due to higher resistance to the flow. However, the velocity improves near the center line of the artery. Further, the fluid's velocity is very high in the constricted (stenotic zone) region.

Computational Analysis of Subcooled Flow Boiling in a Vertical Minichannel with Two Different Shapes under Various Mass Fluxes

In the current research project, two-dimensional numerical simulations are conducted to analyze the effects of geometrical configuration on flow structures and the thermal performances of subcooled flow boiling. The CFD simulations are carried out in two different configurations (straight and periodic constriction expansion) in a minichannel mounted vertically at four mass fluxes (500 kg/m2s; 836.64 kg/m2s; 1170 kg/m2s; and 2535 kg/m2s). The present predicted results exhibit excellent accordance with the previous experiments, with mean errors of 6.39% and 9.78%, demonstrating the efficiency of the present numerical study. The simulation results show that the periodic constriction expansion design provides good mixing between the layers, leading to a 43.11% mean enhancement of the thermal transfer, which is more important than the slight pressure drop penalty of 4.32 for a mass flux of 500 kg/m2s due to the combined pressure drop along the minichannel that resulted from the periodic constriction and expansion regions. Furthermore, the visualization of flow patterns shows that the bubbly flow is the dominant flow regime in the periodic constriction-expansion configuration.

Electromigration at atomic-scale metal nanojunctions driven by “lucky electrons”

We have performed electrical break junction experiments on gold nanocontacts. When the nanocontacts are in the diffusive transport regime, we find that the number of atoms removed by Joule heating is rather small (less than 15%) and that the majority of atoms are removed at voltages determined by the surface self-diffusion potentials of gold. We propose a model in which a small fraction of electrons (“lucky electrons”) traverse the constricted region ballistically and transfer their kinetic energy to metal atoms and remove them. Electromigration experiments on other metal species of high melting temperatures (Ni, Pd) strongly support this interpretation.

Protein Sizing with 15 nm Conical Biological Nanopore YaxAB.

Nanopores are promising single-molecule tools for the electrical identification and sequencing of biomolecules. However, the characterization of proteins, especially in real-time and in complex biological samples, is complicated by the sheer variety of sizes and shapes in the proteome. Here, we introduce a large biological nanopore, YaxAB for folded protein analysis. The 15 nm cis-opening and a 3.5 nm trans-constriction describe a conical shape that allows the characterization of a wide range of proteins. Molecular dynamics showed proteins are captured by the electroosmotic flow, and the overall resistance is largely dominated by the narrow trans constriction region of the nanopore. Conveniently, proteins in the 35-125 kDa range remain trapped within the conical lumen of the nanopore for a time that can be tuned by the external bias. Contrary to cylindrical nanopores, in YaxAB, the current blockade decreases with the size of the trapped protein, as smaller proteins penetrate deeper into the constriction region than larger proteins do. These characteristics are especially useful for characterizing large proteins, as shown for pentameric C-reactive protein (125 kDa), a widely used health indicator, which showed a signal that could be identified in the background of other serum proteins.

Comparative Cytogenetics and Fluorescent Chromosome Banding in Five Indian Species of Dipcadi Medik

The genus Dipcadi Medik. (Subfamily: Scilloideae) has a narrow distribution in India and several overlapping morphological traits make the genus taxonomically challenging at the species level. Cytogenetic characterization can provide additional taxonomic data and can be used to evaluate genetic diversity at the species level. We have accomplished comparative karyotype analysis and fluorescence banding patterns using 4′-6-Diamidino-2-phenylindole (DAPI) and Chromomycin A3 (CMA) in five Indian species for the first time. The karyotypes of D. concanense and D. goaense exhibited similar fluorochrome banding profiles. However, D. montanum, D. ursulae and D. erythraeum differ distinctly in their karyotypes. In all taxa, CMA+ve/DAPI−ve or DAPI0 (GC-rich) constitutive heterochromatin was located at the constriction region or terminal satellite of the nucleolar chromosome. DAPI+ve/CMA−ve or CMA0 (AT-rich) heterochromatin dominates in D. montanum, D. ursulae and D. erythraeum. However, D. erythraeum shows a distinct variation in fluorochrome banding pattern from all other species. The distribution of CMA and DAPI bands is a reflection of heterochromatin composition and variations acquired by different species. This characterization can be used to assess phylogenetic relationships in the understudied genus Dipcadi and may serve as a basis for other genomic analyses and evolutionary studies.

Impact of Stenosis Geometry on Blood Flow Behavior in Human Artery

Atherosclerosis in human arteries affects the blood flow behaviour in different ways and sometimes leads to fatal diseases. This study is focused on the impact of the elasticity of human arteries and the geometry of stenosis on non-Newtonian blood flow across the constricted arteries. The mathematical analysis is carried out using MatLab software. The elastic nature of human arteries and the non-Newtonian nature of blood are described under the power-law, Herschel-Bulkley and Casson model. The numerical derivations for blood flow velocity, volume flow rate and wall shear stress are derived for all three types of the model under consideration and results are also depicted graphically. The influence of elasticity, flow index, yield stress and geometry of stenosis on the blood flow is examined. Power-law model gives higher velocity as compared to Herschel-Bulkley model for the fixed magnitude of flow index 𝑛. It is observed that in the constricted region, the volume flow rate amplifies with the increasing value of stenosis shape parameter 𝑚 for the fixed magnitude of flow index 𝑛. The height of stenosis has a diminishing effect on the flow rate. A comparative analysis of the outcomes of the present study and that of the other published work is carried out for authentication.

The dynamin-related protein Vps1 and the peroxisomal membrane protein Pex27 function together during peroxisome fission.

Dynamin-related proteins (Drp) mediate a variety of membrane remodelling processes. The fungal Drp, Vps1, is required for endocytosis, endosomal sorting, vacuole fusion and peroxisome fission and breakdown. How Drps, and in particular Vps1, can mediate their function at so many different subcellular locations is of interest to our understanding of cellular organisation. We found that the peroxisomal membrane protein Pex27 is specifically required for Vps1-dependent peroxisome fission in proliferating cells but is not required for Dnm1-dependent peroxisome fission. Pex27 accumulates in constricted regions of peroxisomes and affects peroxisome geometry upon overexpression. Moreover, Pex27 physically interacts with Vps1 in vivo and is required for accumulation of a GTPase defective Vps1 mutant (K42A), on peroxisomes. During nitrogen starvation, a condition that halts cell division and induces peroxisome breakdown, Vps1 associates with the pexophagophore. Pex27 is neither required for Vps1 recruitment to the pexophagophore nor for pexophagy. Our study identifies Pex27 as a Vps1 specific partner for the maintenance of peroxisome number in proliferating yeast cells.

Impact of unsteadiness on the non-Newtonian flow of blood in a vascular tube with stenosis and aneurysm: analytical solution

A theoretical analysis is conducted to explore the time-dependent problem of blood flow streaming in a tapered vascular tube having post-stenotic dilatation followed by symmetric arterial stenosis. Blood is considered a homogeneous incompressible fluid whose rheological behavior is estimated by Herschel–Bulkley fluid model. By exploiting the prescribed boundary conditions, unique expressions of velocity, flow rate, plug core radius and resistance to the flow have been made by handling the complexity of the governing equations with the standard perturbation technique. The steady-state component of the pressure gradient originating due to the functioning of the heart is computed numerically. The effects of various parameters involved in the problem in accordance with the stenosis and aneurysm on the different flow variables are elaborated with the help of graphs. It has been investigated that the rheological parameters have considerable effects on the dynamic of the flow throughout the constricted region of the vascular tube.

Discrimination of cationic peptides using malaria translocon, EXP2, nanopores.

Protein identification provides crucial information on the extensive biochemical reaction in our life. Although mass spectrometry has generally been used for protein/peptide identification, the scarcity of proteins in biological samples and post-translational modifications have remained a challenge. Nanopores, which provide rapid and sensitive single-molecule analysis have attracted attention as an alternative to these techniques. The sensing accuracy of nanopores mainly depends on the congeniality of electrostatic affinity and size between the nanopores and molecules. In this study, we evaluated the capability of peptide detection on EXP2 nanopore, which intrinsically transports a polypeptide chain in vivo as a malaria translocon, using peptides with different sizes and numbers of charges. Our previous studies found that wild-typed EXP2 (Wt-EXP2) nanopore could detect and identify two types of poly-L-lysine (short poly-L-lysine: 10,000 Da, long poly-L-lysine: 30,000-70,000 Da) with different molecular weights. Although the Wt-EXP2 nanopore could discriminate the poly-cationic peptides, the inherent current noise of the Wt-EXP2 nanopore in the analyte-free state interfered with more accurate single-molecule detection. This current noise probably owing to the fluctuation of its C-terminus region, so we designed ΔD231-EXP2, a variant with the deletion of the C-terminus region. The result shows that the current noise frequency of ΔD231-EXP2 nanopore decreased by half compared with Wt-EXP2, and we succeeded in discriminating the two types of oligo-arginine (R7X, X=W, G). Furthermore, the capture frequencies of these cationic peptides of the EXP2 nanopore were higher than the α-hemolysin nanopore. ΔD231-EXP2 nanopore also could identify the five types of trypsinized fragments (893.4-1,429 Da) from lysozyme. These results suggest that the EXP2 nanopore may be suitable for cationic peptides detection and identification. We are going to apply another variant EXP2NC (K42D+S46F) whose amino acid in the constriction region is exchanged for more accurate peptide identification.

The impact of pathogenic and artificial mutations on Claudin-5 selectivity from molecular dynamics simulations

Tight-junctions (TJs) are multi-protein complexes between adjacent endothelial or epithelial cells. In the blood-brain-barrier (BBB), they seal the paracellular space and the Claudin-5 (Cldn5) protein forms their backbone. Despite the fundamental role in brain homeostasis, little is known on Cldn5-based TJ assemblies. Different structural models were suggested, with Cldn5 protomers generating paracellular pores that restrict the passage of ions and small molecules. Recently, the first Cldn5 pathogenic mutation, G60R, was identified and shown to induce Cl−-selective channels and Na+ barriers in BBB TJs, providing an excellent opportunity to validate the structural models. Here, we used molecular dynamics to study the permeation of ions and water through two distinct G60R-Cldn5 paracellular architectures. Only the so-called Pore I reproduces the functional modification observed in experiments, displaying a free energy (FE) minimum for Cl− and a barrier for Na+ consistent with anionic selectivity. We also studied the artificial Q57D and Q63D mutations in the constriction region, Q57 being conserved in Cldns except for cation permeable homologs. In both cases, we obtain FE profiles consistent with facilitated passage of cations. Our calculations provide the first in-silico description of a Cldn5 pathogenic mutation, further assessing the TJ Pore I model and yielding new insight on BBB’s paracellular selectivity.

Non-Newtonian characteristics of blood flow in a multi-stenosed elliptical artery: A case of sensitivity analysis

The occurrence and growth of stenosis effectively interrupt the blood flow in the artery, which may result in vascular disease. It makes the study of blood flow in the artery narrowed with crucial stenosis. This work studies the non-Newtonian nature of blood flow in a diseased artery with an elliptic cross-section. The artery is harmed due to several stenosis, which diminishes its lumen. The Phan-Thein–Tanner fluid is considered to analyze the non-Newtonian characteristics of blood. The Phan-Thein–Tanner fluid model is much suitable for blood flow analysis because of its viscoelastic and shear thinning properties. The governing equations are processed to dimensionless form by employing dimensionless variables and assumptions for a mild stenosis case. The solutions of the nondimensional equations are acquired analytically. The visual examination of the exact solutions is discussed in detail. The fluid velocity is strongly affected by stenosis height, and a more significant disorder is generated in the constricted region with the growing size of stenosis. The flow velocity is found as a decreasing function of the Weissenberg number. The velocity profile is parabolic and axisymmetric as well. The most significant and least influential physical constraints are identified by completing the local sensitivity analysis.

Conformational Dynamics of Loop L3 in OmpF: Implications toward Antibiotic Translocation and Voltage Gating.

In the present work, we delineate the molecular mechanism of a bulky antibiotic permeating through a bacterial channel and uncover the role of conformational dynamics of the constriction loop in this process. Using the temperature accelerated sliced sampling approach, we shed light onto the dynamics of the L3 loop, in particular the F118 to S125 segment, at the constriction regions of the OmpF porin. We complement the findings with single channel electrophysiology experiments and applied-field simulations, and we demonstrate the role of hydrogen-bond stabilization in the conformational dynamics of the L3 loop. A molecular mechanism of permeation is put forward wherein charged antibiotics perturb the network of stabilizing hydrogen-bond interactions and induce conformational changes in the L3 segment, thereby aiding the accommodation and permeation of bulky antibiotic molecules across the constriction region. We complement the findings with single channel electrophysiology experiments and demonstrate the importance of the hydrogen-bond stabilization in the conformational dynamics of the L3 loop. The generality of the present observations and experimental results regarding the L3 dynamics enables us to identify this L3 segment as the source of gating. We propose a mechanism of OmpF gating that is in agreement with previous experimental data that showed the noninfluence of cysteine double mutants that tethered the L3 tip to the barrel wall on the OmpF gating behavior. The presence of similar loop stabilization networks in porins of other clinically relevant pathogens suggests that the conformational dynamics of the constriction loop is possibly of general importance in the context of antibiotic permeation through porins.